Shima D T, Saunders K B, Gougos A, D'Amore P A
Laboratory for Surgical Research, Children's Hospital, Boston, MA 02115.
Differentiation. 1995 Feb;58(3):217-26. doi: 10.1046/j.1432-0436.1995.5830217.x.
The endothelium maintains a developmental plasticity which allows rapid phenotypic change in response to extracellular signals during normal processes, such as corpus luteum formation and wound healing, and in pathologic processes, such as tumor angiogenesis. Endothelial cells (EC) in culture have been very useful for investigating various aspects of endothelial growth and behavior. In spite of documented similarities between EC in vitro and the endothelium in vivo, many characteristics of the vessel endothelium are lost when the cells are placed into culture. We have undertaken to identify differences in gene expression between differentiated vessel endothelium and dedifferentiated EC. We utilized a new technique called differential display which compares polymerase chain reaction (PCR)-amplified mRNA from two (or more) cell populations. Endothelium scraped directly from freshly obtained aortas, and demonstrated to be free of contaminants, were used as the source of differentiated RNA, whereas proliferating, primary explanted EC grown for five days in the presence of basic fibroblast growth factor (bFGF) provided a pool of 'dedifferentiated' RNA. Using differential display, we have observed numerous reproducible differences in gene expression. To confirm that the expression differences visualized by differential display represented actual differences in gene expression, we isolated vessel-specific and culture-specific cDNA tags for additional analysis. Three cDNA tags specific to vessel endothelium were cloned and sequenced, and compared to nucleotide and protein databases. Two of the clones (A1 and 2.5) displayed no significant sequence similarity, whereas a third clone (A2) is nearly identical to a human expressed sequence tag (EST) and has significant sequence similarities to a plant and Xenopus ubiquitin-like protein. Northern and/or in situ hybridization analysis of the A1 and A2 genes confirmed their restricted expression to the vessel endothelium. The expression of A1 by the endothelium in vivo is not simply a function of growth state, as cultured cells did not express A1 even when grown to postconfluence. One other cDNA fragment, selected as a culture-induced gene, was identified by sequence analysis as the bovine homologue of laminin B1, and Northern analysis confirmed that expression was induced upon culturing of EC. Use of differential display to study endothelial gene expression will allow us to investigate the molecular mechanisms that underlie initiation and maintenance of endothelial differentiation.
内皮细胞具有发育可塑性，在正常生理过程（如黄体形成和伤口愈合）以及病理过程（如肿瘤血管生成）中，能响应细胞外信号而快速发生表型变化。培养的内皮细胞（EC）对于研究内皮细胞生长和行为的各个方面非常有用。尽管有文献记载体外培养的EC与体内内皮细胞存在相似之处，但当细胞置于培养环境中时，血管内皮细胞的许多特性会丧失。我们致力于鉴定分化的血管内皮细胞与去分化的EC之间基因表达的差异。我们采用了一种名为差异显示的新技术，该技术可比较来自两个（或更多）细胞群体的聚合酶链反应（PCR）扩增的mRNA。直接从新鲜获取的主动脉刮取且经证实无污染物的内皮细胞，用作分化RNA的来源，而在碱性成纤维细胞生长因子（bFGF）存在下培养五天的增殖性原代外植体EC则提供了一组“去分化”RNA。通过差异显示，我们观察到了许多可重复的基因表达差异。为了证实差异显示所观察到的表达差异代表基因表达的实际差异，我们分离了血管特异性和培养特异性的cDNA标签用于进一步分析。克隆并测序了三个血管内皮细胞特异性的cDNA标签，并与核苷酸和蛋白质数据库进行比较。其中两个克隆（A1和2.5）未显示出明显的序列相似性，而第三个克隆（A2）与人类表达序列标签（EST）几乎相同，并且与一种植物和非洲爪蟾泛素样蛋白具有显著的序列相似性。对A1和A2基因的Northern和/或原位杂交分析证实它们在内皮细胞中特异性表达。体内内皮细胞对A1的表达并非简单地取决于生长状态，因为培养的细胞即使生长至汇合后也不表达A1。另一个被选为培养诱导基因的cDNA片段，经序列分析鉴定为层粘连蛋白B1的牛同源物，Northern分析证实EC培养后该基因表达被诱导。使用差异显示技术研究内皮细胞基因表达将使我们能够探究内皮细胞分化起始和维持的分子机制。
